A variety of materials (i.e., cationic lipids, polymers: natural and synthetic and peptides) have been utilized to fabricate non-viral delivery systems (M. J. Tiera et al, Curr. Gene Ther. 6 (2006) 59-71,13) which have several advantages in terms of safety, ease of preparation, reproducibility, ability to carry large nucleic acid constructs and stability (X. Gao et al, AAPS J. 9 (2007) E92-E104). Cationic lipids and cationic polymers for gene delivery may cause toxic effect in vitro and in vivo. For example lipoplexes caused several changes to cells, which included cell shrinking, reduced number of mitoses and vacuolization of the cytoplasm (K. Lappalainen et al, Pharm. Res. 11 (1994) 1127-1131). Cationic polymers, viz., polyethylenimine (PEI), polyamidoamine (PAMAM), polypropylenimine (PPI), poly-L-lysine (PLL), cationic dextran, polyallylamine (PAA), dextran-oligoamine based conjugates and chitosan, (M. D. Brown et al, Int. J. Pharm. 229 (2001) 1-21; H. Hosseinkhani et al, Gene Ther. 11 (2004) 194-203.), are amongst the preferable materials for the preparation of non-viral vectors in terms of their long-term safety and biocompatibility. PLL, PAA and many others were abandoned due to its low transfection efficiency and higher cytotoxicity. Dextran-oligoamine based transfection in a wide range of cell lines is very low in comparison to other cationic vectors based on PEI, dendrimers etc. Of these, PEI is one of the most successful and widely studied gene delivery polymers due to its membrane destabilization potential, high charge density (DNA condensation capability) and ability to protect endocytosed DNA from enzymatic degradation, thus perform DNA transfer efficiently into the cells (U. Lungwitz et al, Eur. J. Pharm. Biopharm. 60 (2005) 247-266). Branched PEI contains primary, secondary and tertiary amines in a ratio of 1:2:1 with pKa values spanning around the physiological pH, providing remarkable buffering capacity. The primary amines are mainly responsible for high degree of DNA binding, but contribute maximum toxicity during transfection, while the secondary and tertiary amino groups provide good buffering capacity to the system (X. Gao et al, Mol. Ther. 11 (2005) S427-S428). Though high charge density of the system increases the transfection efficiency, it simultaneously contributes to increased cytotoxicity. Actually, the efficacy of the transfection system is a sweet compromise between the transfection efficiency and the cytotoxicity. Several research groups have attempted to reduce the charge associated toxicity of PEI either by incorporating various ligands like peptides, polyethylene glycols, polysaccharides or by substituting the amines with imidazolyl and acyl functions (U. Lungwitz et al, Eur. J. Pharm. Biopharm. 60 (2005) 247-266; S. Patnaik et al, J. Control. Release 114 (2006) 398-409; A. Swami et al, Int. J. Pharm. 335 (2007) 180-192; S, Nimesh et al, Int. J. Pharm. 337 (2007) 265-274). However, these modifications result in partial blockage of the net amino charge on PEI, thus making a compromise on transfection efficiency. These ligands react with amines of PEI by shielding the charge which is responsible for the cytotoxicity of PEI as well as the DNA binding ability and in turn the transfection efficiency of PEI, thus making a compromise in a way.
There is a need to design a system based on cationic polymers where, primary amino content could be kept to minimum and secondary, tertiary and quaternary content could be increased to obviate the drawbacks listed above and reduce cytotoxicity.
In the present invention, in order to address the problem of cytoxicity in PEI and to improve upon the transfection efficiency, PEI nanoparticles have been prepared using crosslinkers such as, 1,4-butanediol digycidyl ether (bisepoxide) and 1,6-hexandial (bisaldehyde) which reduces the charge dependent toxicity of native PEI (25 kDa).
The novelty of the invention is in the crosslinkings which do not alter the net charge of the PEI system, rather it partially converts the primary amines (main source of toxicity) to secondary, the secondary to tertiary amines (which play important role in buffering capacity) and/or tertiary to quaternary amines making the highly toxic PEI into transfectionally competent system.
Another novelty of the present invention is in providing nanoparticle based efficient polymeric transfection agent by converting the amine moiety of the cationic polymer from primary to secondary to tertiary and/or further to quaternary state.